Method of combination therapy using anti-c-met antibody and sorafenib

ABSTRACT

Provided is a method of combination therapy for prevention or treatment of a cancer including or consisting essentially of co-administering sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof to a subject. The method of combination therapy can achieve an excellent synergistic effect and lower the effective dose of the anti-c-Met antibody, thereby enabling a more effective cancer treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2013-0035292 filed on Apr. 1, 2013, in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 137,012 Byte ASCII (Text) file named “715843SequenceListing.TXT,” created on Apr. 1, 2014.

BACKGROUND

1. Field

Provided is a method of combination therapy for prevention and/or treatment of a cancer, including co-administering sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof to a subject in need thereof.

2. Description of the Related Art

c-Met, a typical receptor tyrosine kinase (RTK) present at the surface of cells, binds to its ligand, hepatocyte growth factor (HGF) to promote intracellular signal transduction thereby not only promoting the growth of cells but also being over-expressed in cancer cells so that it is widely implicated in cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, and the like. For these reasons, c-Met has been emerging as an important target for cancer treatment.

There have been many studies on the possibility of c-Met as a new target for cancer treatment, and several drugs relating to c-Met have been developed and subjected to clinical trials. However, most of c-Met inhibitors cannot sufficiently exhibit their effects due to cross-talk of a downstream signaling pathway and other signaling pathways and feedback effects of a downstream signaling pathway and other receptor tyrosine kinases. Therefore, as a new way for inhibiting c-Met, a combined therapy with other pre-existing drugs has been sought.

Accordingly, there is a desire for the development of an efficient combination therapy using an anti-c-Met antibody and a drug targeting another tumor-related protein.

SUMMARY

It is suggested that the combined therapy of an anti-c-Met antibody and sorafenib could achieve significant synergy effects in treating a cancer.

Accordingly, one embodiment provides a method of combined therapy for prevention and/or treatment of a cancer including or consisting essentially of co-administering sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof to a subject in need of prevention and/or treatment of the cancer. In a particular embodiment, the anti-c-Met antibody or antigen-binding fragment thereof specifically binds to an epitope including 5 or more amino acids (e.g., contiguous amino acids) within the SEMA domain (e.g., SEQ ID NO: 79) of c-Met protein.

Another embodiment provides sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof for use of combined therapy for prevention and/or treatment of a cancer.

Another embodiment provides a pharmaceutical composition for combined therapy for prevention and/or treatment of a cancer, including or consisting essentially of sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof as active ingredients.

Still another embodiment provides a kit for prevention and/or treatment of a cancer, including a first pharmaceutical composition including or consisting essentially of sorafenib as an active ingredient, a second pharmaceutical composition including or consisting essentially of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient, and a package container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing cell viabilities depending on concentrations of anti-c-Met antibody in MKN45 gastric cancer cell when the anti-c-Met antibody is administered alone or together with sorafenib.

FIG. 2 is a graph showing cell viabilities depending on concentrations of anti-c-Met antibody in EBC1 lung cancer cell when the anti-c-Met antibody is administered alone or together with sorafenib.

FIG. 3 is a graph showing cell viabilities depending on concentrations of anti-c-Met antibody in HT29 colon cancer cell when the anti-c-Met antibody is administered alone or together with sorafenib.

FIG. 4 is a graph showing cell viabilities depending on concentrations of anti-c-Met antibody in HCC1806 breast cancer cell when the anti-c-Met antibody is administered alone or together with sorafenib.

FIG. 5 is a graph showing tumor volume in a tumor xenograft model of gastric cancer cell line MKN45 with time (n=10).

FIG. 6 is a graph showing tumor volume in a tumor xenograft model of colon cancer cell line HT29 with time (n=10).

DETAILED DESCRIPTION

Targeting anticancer drugs having specific targets, such as antibodies, tend to have acquired resistances compared to non-targeting anticancer drugs. In order to avoid acquired resistances resulting from administration of such targeting anticancer drugs, co-inhibition of a factor that induces resistance to the anticancer drug, thereby maximizing the efficacies of the anticancer drug, is suggested. In addition, many targeting anticancer drugs have limited drug coverage, and it is possible to extend the coverage of the targeting anticancer drug by co-administration (combined therapy) with an inhibitor against other factor. Furthermore, in the case of targeting anticancer drugs with acquired resistances as well as with efficacies, it is possible to improve the efficacies of the targeting anticancer drugs by co-administration with other active drugs, thereby decreasing their dose. Accordingly, by such co-administration, a decrease in the dose of the targeting anticancer drugs administered can be achieved, thereby increasing the efficacies thereof, and minimizing toxicities, which affect on internal organs.

The invention provides a combined therapy by co-administration of an anti-c-Met antibody, which is a targeting anticancer drug, and another anticancer drug. Such combined therapy can create a synergistic effect and can improve the efficacy of the anti-c-Met antibody, thereby effectively decreasing the required dose of the anti-c-Met antibody. The decreased dose of the anti-c-Met antibody can lead to minimized side effects and maximized anticancer efficacies. In addition, by the combined therapy, an anti-c-Met antibody can exhibit an anticancer effect even in cancers in which the anti-c-Met antibody otherwise would exhibit no anticancer effect or only marginal effect when administered alone. Furthermore, a resistance to the anti-c-Met antibody can be overcome. The cancer, in which an anti-c-Met antibody exhibits no anticancer effects when administered alone, has innate resistance to the anti-c-Met antibody, and the innate resistance to the anti-c-Met antibody can be overcome by the combined therapy, allowing the extension of the coverage of the anti-c-Met antibody.

In particular, the invention provides a combined therapy by co-administration of an anti-c-Met antibody and sorafenib. More particularly, provided is a combined therapy to inhibit the activity of HGF/c-Met, which is an important growth factor of cancer cells, and the activity of vascular endothelial growth factor (VEGF), which is an oncogenic factor, and/or to block a downstream signaling pathway of c-Met, such as RAS/RAF/MEK/ERK pathway, thereby achieving a synergistic anticancer effect and decreasing the effective dose of each drug.

Although VEGF is present in normal cells, it is particularly secreted from cancer cells, and binds to VEGF receptor (VEGFR), leading to angiogenesis. Cancer cells are supplied with nutrients necessary for their growth through the newly formed blood vessel. Overexpression of VEGF serves as a cause of various diseases and implicated in poor prognosis of cancer, such as cancer invasion or metastasis as well as cancer development. In addition, the RAS/RAF/MEK/ERK pathway is a signaling pathway from a receptor, such as c-Met, on a cell surface into DNA in nucleus, and an abnormal regulation of the pathway acts as a cause to induce a cancer. Therefore, an increased anticancer effect can be obtained by co-administration of an anti-c-Met antibody and a drug inhibiting at least one (e.g., both) of a downstream signaling pathway of c-Met (e.g., RAS/RAF/MEK/ERK pathway) and VEGF receptor.

Accordingly, in one embodiment, sorafenib, which is a multi-kinase inhibitor, is selected as an inhibitor of both the VEGF receptor and RAS/RAF/MEK/ERK pathway, and co-administered with an anti-c-Met antibody, achieving an increased efficacy of the antibody and synergistic effects. In particular, the growth of cancer cells can be more effectively inhibited by simultaneously regulating the two intracellular signaling pathways by co-administration of an anti-c-Met antibody and sorafenib.

The co-administration of an anti-c-Met antibody and sorafenib leads to cancer cell growth inhibition by targeting c-Met, thereby blocking the c-Met signaling pathway and the downstream RAS/RAF/MEK/ERK pathway at the same time; and by suppressing angiogenesis by VEGF, thereby blocking the supply of nutrients necessary for the growth of the cancer cells. Cancer cell growth is more effectively inhibited by the co-administration of an anti-c-Met antibody and sorafenib as compared to a single administration of each drug. In addition, the co-administration of an anti-c-Met antibody and sorafenib leads to decreased side effects, such as agonism, and increased anticancer effects. Thus, the co-administration of an anti-c-Met antibody and sorafenib exhibits an excellent anticancer effect even at a low dose of each drug, and/or even in a cancer for which the anti-c-Met antibody alone exhibits no anticancer effect.

Accordingly, one embodiment provides a method of combined therapy (co-administration) for prevention and/or treatment of a cancer including (or consisting essentially of, or consisting of) co-administering sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof, to a subject in need of prevention and/or treatment of the cancer. The sorafenib and the anti-c-Met antibody or antigen-binding fragment thereof may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The method may further include a step of identifying a subject who is in need of the prevention and/or treatment of a cancer, prior to the co-administration step. The step of identifying may be conducted by any manners and/or methods known to relevant field for identifying whether or not a subject needs the prevention and/or treatment of cancer. For example, the step of identifying may include diagnosing a subject as a cancer patient having a cancer, or identifying a subject who is diagnosed as a cancer patient.

In one concrete embodiment, the co-administration may be conducted by administering a mixed formulation of sorafenib and an anti-c-Met antibody or antigen-binding fragment thereof, as described herein. In another concrete embodiment, the co-administration may be conducted by a first step of administering sorafenib, and a second step of administering an anti-c-Met antibody or antigen-binding fragment thereof, wherein the first and the second administration steps may be conducted simultaneously or sequentially. In case of the sequential administration, the first step and the second step may be performed in any order. The sorafenib and anti-c-Met antibody or antigen-binding fragment thereof may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher.

The subject may be selected from mammals including primates, such as humans and monkeys, and rodents, such as mice and rats. Furthermore, the subject may be a cancer subject, or subjects having resistance to an anti-c-Met antibody. Hence, the prevention and/or treatment method may further include a step of identifying a subject having resistance to an anti-c-Met antibody, prior to the administration step.

Another embodiment provides a use of anti-c-Met antibody and sorafenib for combined therapy for treatment and/or prevention of a cancer.

Another embodiment provides a pharmaceutical composition for combined therapy for prevention and/or treatment of a cancer including or consisting essentially of sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof as active ingredients.

The pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition including or consisting essentially of two or more active ingredients) of sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof. Sorafenib and the anti-c-Met antibody or antigen-binding fragment thereof can be present in any amount that is pharmaceutically effective when used together. The composition thus formulated can be used for simultaneous administration of the two active ingredients.

Alternatively, sorafenib and the anti-c-Met antibody or antigen-binding fragment thereof can each be formulated in a separate composition, and the two active ingredients can be separately administered simultaneously or sequentially. For instance, a first pharmaceutical composition including or consisting essentially of a pharmaceutically effective amount of sorafenib as an active ingredient and a second pharmaceutical composition including or consisting essentially of a pharmaceutically effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient can be administered simultaneously or sequentially. In the case of the sequential administration, any order of administration may be used.

In another embodiment, a kit for prevention and/or treatment of a cancer is provided, wherein the kit includes (a) a first pharmaceutical composition including or consisting essentially of sorafenib as an active ingredient, (b) a second pharmaceutical composition including or consisting essentially of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient, and (c) a package container. Sorafenib and the anti-c-Met antibody or an antigen-binding fragment thereof may be used in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The package container can be any container that holds or otherwise links the two compositions in individual containers together in a single unit (e.g., a box that holds both containers, or plastic wrap that binds both containers together), or the package container may be a single, divided container having at least two chambers that each hold one of the two compositions.

The combined therapy of sorafenib and an anti-c-Met antibody or an antigen-binding fragment thereof can achieve excellent synergistic effects and decreased dose when compared to single administration of each drug. In addition, the combined therapy can maintain an excellent anticancer effect, even when the administration interval gets longer. Furthermore, the combined therapy can exhibit anticancer effects on a cancer having agonism against an anti-c-Met antibody and/or a cancer on which an anti-c-Met antibody exhibits no effect or only insignificant effect.

Sorafenib (Trade Name: Nexavar; IUPAC Name: 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide; CAS Registry Number 284461-73-0), which is a medicament approved by FDA in 2005, is used for the treatment of kidney cancer, liver cancer, and the like, and has the following chemical structure:

Sorafenib is a multi-kinase inhibitor which inhibits VEGFR, platelet-derived growth factor receptor (PDGFR), and/or RAF kinases, etc.

To verify the increase in efficacy of an anti-c-Met antibody by co-administration of such a multi-kinase inhibitor (sorafenib) and anti-c-Met antibody, the synergistic effect by the co-administration to cancer cells in which the anti-c-Met antibody exhibits an anticancer effect, such as gastric cancer cells, lung cancer cells, and the like, was measured (see Examples 1 and 2). The effect of the co-administration to cancer cells in which the anti-c-Met antibody exhibits no anticancer effect when administered alone, such as colon cancer cells, breast cancer cells, and the like, was also measured (see Examples 3 and 4). As the result, in cancer cells in which the anti-c-Met antibody exhibits an anticancer effect, a synergistic effect was observed by the co-administration compared to a single administration of the anti-c-Met antibody only (see Examples 1 and 2). In addition, in cancer cells in which the anti-c-Met antibody exhibits no anticancer effect when administered alone, an anticancer effect by the anti-c-Met antibody was acquired by the co-administration of the anti-c-Met antibody with sorafenib (see Examples 3 and 4). These results indicate that the co-administration can achieve not only excellent synergistic effects, but also improvement of the efficacy of an anti-c-Met antibody and overcoming of resistance to the anti-c-Met antibody, thereby exhibiting an excellent effect even on the cancer cells where the anti-c-Met antibody exhibits no effect and thus has therapeutic limitations. This allows the extension of the coverage of the anti-c-Met antibody and decreases side effects of the anti-c-Met antibody by lowering the effective dose thereof.

“c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF). c-Met may be derived (obtained) from any species, particularly a mammal, for instance, primates such as human c-Met (e.g., GenBank Accession No. NP_(—)000236), monkey c-Met (e.g., Macaca mulatta, GenBank Accession No. NP_(—)001162100), or rodents such as mouse c-Met (e.g., GenBank Accession No. NP_(—)032617.2), rat c-Met (e.g., GenBank Accession No. NP_(—)113705.1), and the like. The c-Met protein may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession No. NM_(—)000245, a polypeptide having the amino acid sequence identified as GenBank Accession No. NP_(—)000236 or extracellular domains thereof. The receptor tyrosine kinase c-Met participates in various mechanisms, such as cancer incidence, metastasis, migration of cancer cells, invasion of cancer cells, angiogenesis, and the like.

The anti-c-Met antibody may be any antibody targeting c-Met protein and inhibiting the activity thereof.

The antigen-binding fragment of the anti-c-Met antibody may refer to a fragment including an antigen binding region of the anti-c-Met antibody, and can be selected from the group consisting of a complementarity determining region (CDR), fragment including CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂ of the anti-c-Met antibody. The anti-c-Met antibody may also include a variant of the antibody. The variant of the antibody may be any isotype of antibodies derived from human and other animals found in nature and/or one including any Fc region of antibodies derived from human and other animals, having a mutated hinge wherein at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid is changed, deleted, inserted, or added. Unless stated otherwise, the anti-c-Met antibody may include the variants of the antibody as well as the antibody with no variation.

The anti-c-Met antibody may recognize a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope. It may be any antibody or antigen-binding fragment that acts on c-Met to induce c-Met intracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and includes a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin identity/homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region having the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79), is a loop region between the second and the third propellers within the epitopes of the SEMA domain. This region acts as an epitope for the anti-c-Met antibody provided in the present invention.

The term “epitope,” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more contiguous (consecutive on primary, secondary (two-dimensional), or tertiary (three-dimensional) structure) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide having 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide includes the amino sequence of SEQ ID NO: 73 (EEPSQ) which serves as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope having the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope having the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which includes 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may include or consist essentially of:

(i) at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence including 8-19 consecutive amino acids within the amino acid sequence of SEQ ID NO: 2 including amino acid residues from the 3^(rd) to 10^(th) positions of the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence including 6-13 consecutive amino acids within the amino acid sequence of SEQ ID NO: 85 including amino acid residues from the 1^(st) to 6^(th) positions of the amino acid sequence of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

(ii) at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence comprising 9-17 consecutive amino acids within the amino acid sequence of SEQ ID NO: 89 including amino acid residues from the 1^(st) to 9^(th) positions of the amino acid sequence of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;

(iii) a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

(iv) a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas Ito VI, below:

Formula I: Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser (SEQ ID NO: 4), wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II: Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr (SEQ ID NO: 5), wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III: Asp-Asn-Trp-Leu-Xaa₆-Tyr (SEQ ID NO: 6), wherein Xaa₆ is Ser or Thr,

Formula IV: Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala (SEQ ID NO: 7), wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Formula V: Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃ (SEQ ID NO: 8), wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI: Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr (SEQ ID NO: 9), wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₈ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment may include or essentially consist of (i) a heavy variable region including a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and (ii) a light variable region including a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR), which serve an important role in antigen binding in variable regions of chimeric antibodies, into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained and, thus, application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be a form isolated (or separated) from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. In a particular embodiment, the antigen-binding fragment may be scFv, (scFv)₂, scFvFc, Fab, Fab′, or F(ab′)₂, but is not limited thereto.

Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment.

Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be the same as described in the above, for example, those including the amino acid length of 1 to 100, 2 to 50, particularly 5 to 25, and any kinds of amino acids may be included without any restrictions.

The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region having the amino acid sequence of SEQ ID NO: 100 (U7-HC6), 101 (U6-HC7), 102 (U3-HC9), 103 (U6-HC8), or 104 (U8-HC5), or a hinge region having the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). In particular, the hinge region comprises the amino acid sequence of SEQ ID NO: 100 or 101.

In one embodiment of the anti-c-Met antibody or antigen-binding fragment, the variable domain of the heavy chain includes the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and the variable domain of the light chain includes the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, under Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the disclosure of which is incorporated in its entirety herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

In the anti-c-Met antibody, the portion of the light chain and the heavy chain portion excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).

By way of further example, the anti-c-Met antibody or the antibody fragment may include or consisting essentially of:

(i) a heavy chain including an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66; and

(ii) a light chain including an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

(a) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(b) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(c) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66, and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(d) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(e) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(f) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66, and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(g) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain including the amino acid sequence of SEQ ID NO: 108;

(h) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain including the amino acid sequence of SEQ ID NO: 108; and

(i) an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66, and a light chain including the amino acid sequence of SEQ ID NO: 108.

According to an embodiment, the anti-c-Met antibody may include or consist essentially of a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66 and a light chain including the sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68, or a heavy chain comprising the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66 and a light chain including the sequence of SEQ ID NO: 108. The composition including an antibody or an antigen-binding fragment can be formulated into immunoliposomes. Additionally, the pharmaceutical composition or the combined mixture may be formulated into immunoliposomes. Liposomes including an antibody may be prepared using methods that are well-known in the art. The immunoliposomes may be produced from a lipid composition including phosphatidylcholine, cholesterol, and PEGylated phosphatidylethanolamine by reverse-phase evaporation. In a particular example, Fab′ may be conjugated to liposomes by disulfide reformation. The liposome may further include an anticancer agent such as doxorubicin.

The polypeptide with the amino acid sequence of SEQ ID NO: 70 is a light chain including the human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of the amino acid sequence of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of the amino acid sequence of SEQ ID NO: 68; positioned within CDR-L1) with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibit increased activities, such as c-Met binding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti-c-Met antibody may include or consist essentially of a light chain complementarity determining region including the amino acid sequence of SEQ ID NO: 106, a light chain variable region including the amino acid sequence of SEQ ID NO: 107, or a light chain including the amino acid sequence of SEQ ID NO: 108.

In another embodiment, the anti-c-Met antibody may be an antibody including or consisting essentially of a heavy chain including the amino acid sequence of SEQ ID NO: 109 and a light chain including the amino acid sequence of SEQ ID NO: 110, or an antigen-binding fragment thereof.

The mixture where a pharmaceutically effective amount of sorafenib and a pharmaceutically effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof are mixed, the first pharmaceutical composition including a pharmaceutically effective amount of sorafenib as an active ingredient, and the second pharmaceutical composition including a pharmaceutically effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient, may be provided (or administered) along with a pharmaceutically acceptable carrier, diluent, and/or excipient.

The pharmaceutically acceptable carrier to be included in the mixture or the pharmaceutical composition may be those commonly used for the formulation of antibodies, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, preservative, and the like.

The pharmaceutical composition, the mixture, or each active ingredient may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

The term “the pharmaceutically effective amount” as used in this specification refers to an amount of which each active ingredient can exert pharmaceutically significant effects.

For one-time administration, a pharmaceutically effective amount of sorafenib and a pharmaceutically effective amount of the anti-c-Met antibodies or antigen binding fragments thereof may be prescribed in a variety of ways, depending on many factors comprising formulation methods, administration manners, ages of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the effective amount of sorafenib may be, but not limited to, in ranges of 0.001 to 100 mg/kg (e.g., 0.001 mg/kg, 0.01 mg/kg, 1 mg/kg, or 10 mg/kg), or 0.02 to 10 mg/kg (e.g., 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, or 5 mg/kg) for one-time administration and the effective amount of the anti-c-Met antibodies or antigen binding fragments thereof may be, but not limited to, in ranges of 0.001 to 100 mg/kg (e.g., 0.001 mg/kg, 0.01 mg/kg, 1 mg/kg, or 10 mg/kg), or 0.02 to 10 mg/kg (e.g., 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, or 5 mg/kg for their one-time administration.

The effective amount for one-time administration may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. For the kit, the effective amount of sorafenib and the effective amount of the anti-c-Met antibodies or antigen binding fragments thereof for one-time administration (single dose) may be contained in a package container as a base unit.

The administration interval between the administrations is defined as a period between the first administration and the following administration. The administration interval may be, but is not limited to, 24 hours to 30 days (e.g., 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6, days, 7 days, 10 days, 14 days, 21 days, or 28 days) and particularly 7 to 14 days or so. For the combined therapy, the first pharmaceutical composition comprising a pharmaceutically effective amount of sorafenib as an active ingredient, and the second pharmaceutical composition comprising a pharmaceutically effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient may be co-administered in a given time interval (e.g., several minutes, several hours or several days, or several weeks) to be determined by the type of disease, a subject's conditions, etc. For example, the first pharmaceutical composition and the second pharmaceutical composition may be simultaneously administered (administration interval within 1 minute) or sequentially administered (administration interval of 1 minute or over), and in case of sequential administration, the administration interval between the first pharmaceutical composition and the second pharmaceutical composition may be 1 to 60 minutes (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or 55 minutes), particularly, 1 minute to 10 minutes, and their administration order may be reversed.

The combined mixture or the pharmaceutical compositions may be a solution in oil or an aqueous medium, a suspension, a syrup, an emulsifying solution form, or they may be formulated into a form of an extract, elixirs, powders, granules, a tablet or a capsule, and they may further include a dispersing agent or a stabilizing agent for their formulation.

In particular, the pharmaceutical composition including the anti-c-Met antibody or antigen binding fragments thereof may be formulated into an immunoliposome. A liposome including an antibody may be prepared using any methods well known in the pertinent field. The immunoliposome may be a lipid composition comprising phosphatidylcholine, cholesterol, and polyethyleneglycol-derived phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may further be included in the liposome.

The pharmaceutical compositions or the method may be used for the prevention and/or treatment of a cancer. The cancer may be a solid cancer or a blood cancer. The cancer may be associated with overexpression and/or abnormal activation of c-Met. Particularly, the cancer may be at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, and the like, but not be limited thereto. The cancer may include a metastatic cancer as well as a primary cancer.

The prevention and/or treatment effects of the cancers may include effects of not only suppressing the growth of the cancer cells but also suppressing deterioration of cancers due to migration, invasion, and metastasis thereof. Therefore, the curable cancers by the combined therapy of the invention include both primary cancers and metastatic cancers.

The combined therapy by co-administration of an anti-c-Met antibody and sorafenib as suggested herein has the following effects: 1) an excellent synergistic effect, 2) extension of the coverage of anti-c-Met antibody to the cancer cells on which the anti-c-Met antibody has no anticancer effect, 3) decrease in side effects of drugs by lowering their effective doses, and the like.

Hereafter, the present invention will be described in detail by examples.

The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

EXAMPLES Reference Example 1 Construction of an Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of a Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of a Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, under Accession No. KCLRF-BP-00220 according to the Budapest Treaty (see Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of a Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with an filter (Amicon). The antibody was stored in PBS before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Reference Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL. After 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in a 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has an identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDRs of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has an identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a BLAST search. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→1), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL. After 24 hours, when the cell number reached 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in a 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of an scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS  (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY  (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA  (SEQ ID NO: 10) CDR-L2 WASTRVS  (SEQ ID NO: 11) CDR-L3 QQSYSAPLT  (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of an Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2 Clone Library constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1  CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2  CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3  CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment comprising L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment comprising L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL. After 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in a 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of (a) a heavy chain comprising the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and (b) a light chain comprising the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of (a) a heavy chain comprising a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and (b) a light chain comprising the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of (a) a heavy chain comprising the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and (b) a light chain comprising the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, (a) a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, (b) a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, (c) a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and (d) a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL. After 24 hours, when the cell number reached 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in a 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was representatively selected for the following examples, and referred as L3-1Y.

Example 1 Verification of Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Gastric Cancer Cell Line

The effect of co-administration of sorafenib and anti-c-Met antibody L3-1Y prepared in Reference Example 1 was verified in a gastric cancer cell line (MKN45 cell line).

In particular, RPMI1640 medium (GIBCO) containing 10% FBS was added to a 96 well plate. MKN45 gastric cancer cells (JCRB, JCRB0254) were seeded thereon in the amount of 5,000 cells/well, and incubated overnight at 37° C. After 24 hours (the next day), the incubated cells were treated with (a) L3-1Y (Reference Example 1) alone or (b) L3-1Y and sorafenib. For the single treatment of L3-1Y alone, the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL. For the co-administration of L3-1Y and sorafenib, the concentration of sorafenib was fixed at 4 μM, which is a concentration of IC₃₀ in MKN45 cell line, and the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL.

At 72 hours after the single treatment or the co-treatment, 100 μL of CellTiter Glo solution (Promega, G7572) was added to each well, and left for 30 minutes at room temperature. The cell number was counted by emission signal, and the emission signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer).

The obtained results are shown in FIG. 1. As shown in FIG. 1, in the MKN45 cell line, the cell growth inhibiting effect is observed even by the single treatment of L3-1Y; however, a more increased cell growth inhibition rate is observed by the co-treatment of L3-1Y and sorafenib, and the increased cell growth inhibiting effect is generally increased depending on the concentration of L3-1Y. In addition, in the MKN45 cell line, the treatment of 0.08 μg/mL of L3-1Y leads to only a little effect of cell growth inhibition, whereas the co-treatment of sorafenib and L3-1Y leads to the cell growth inhibition rate of 50%. Even if it is considered that the single treatment of sorafenib (0 μg/mL of L3-1Y) results in the cell growth inhibition rate of 30%, the synergistic effect caused by the co-treatment of L3-1Y and sorafenib is considerably beyond the expected scope. In particular, the cell growth inhibiting effect obtained by the co-treatment of L3-1Y and sorafenib is more excellent compared to that obtained by the single treatment of 2 μg/mL of L3-1Y, even when the concentration of L3-1Y in the co-treatment is as low as 0.08 μg/mL. These results indicate that by the co-treatment of L3-1Y and sorafenib, the cancer cell growth inhibiting effect of L3-1Y can be considerably increased and the effective dose of L3-1Y can be lowered by 1/25 or lower.

Example 2 Verification of Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Lung Cancer Cell Line

The effect of co-administration of sorafenib and anti-c-Met antibody L3-1Y prepared in Reference Example 1 was verified in a lung cancer cell line (EBC1 cell line).

In particular, RPMI1640 medium (GIBCO) containing 10% FBS added to a 96 well plate. EBC1 lung cancer cells (JCRB, JCRB0820) were seeded thereon in the amount of 5,000 cells/well, and incubated overnight at 37° C. After 24 hours (the next day), the incubated cells were treated with (a) L3-1Y (Reference Example 1) alone or (b) L3-1Y and sorafenib. For the single treatment of L3-1Y alone, the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL. For the co-treatment of L3-1Y and sorafenib, the concentration of sorafenib was fixed at 4 μM, and the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/m L.

At 72 hours after the single treatment or the co-treatment, 100 μL of CellTiter Glo solution (Promega, G7572) was added to each well, and left for 30 minutes at room temperature. The cell number was counted by emission signal, and the emission signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer).

The obtained results are shown in FIG. 2. As shown in FIG. 2, in the EBC1 cell line, the cell growth inhibiting effect is observed even by the single treatment of L3-1Y; however, a more increased cell growth inhibition rate is observed by the co-treatment of L3-1Y and sorafenib, and the increased cell growth inhibiting effect is generally increased depending on the concentration of L3-1Y. In addition, considering that the single treatment of 4 μM of sorafenib (0 μg/mL of L3-1Y) leads to no cell growth inhibiting effect on EBC1 cells, the synergistic effect caused by the co-treatment of L3-1Y and sorafenib is considerably beyond the expected scope. In particular, the cell growth inhibiting effect obtained by the co-treatment of L3-1Y and sorafenib is more excellent compared to that obtained by the single treatment of 2 μg/mL of L3-1Y, even when the concentration of L3-1Y in the co-treatment is as low as 0.08 μg/mL. These results indicate that by the co-treatment of L3-1Y and sorafenib, the cancer cell growth inhibiting effect of L3-1Y can be considerably increased and the effective dose of L3-1Y can be lowered by 1/25 or lower.

Example 3 Verification of the Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Colon Cancer Cell Line

The effect of co-administration of sorafenib and anti-c-Met antibody L3-1Y prepared in Reference Example 1 was verified in a colon cancer cell line (HT29 cell line).

In particular, RPMI1640 medium (GIBCO) containing 10% FBS was added to a 96 well plate. HT29 colon cancer cells (ATCC, HTB-38) were seeded thereon in the amount of 5,000 cells/well, and incubated overnight at 37° C. After 24 hours (the next day), the incubated cells were treated with (a) L3-1Y (Reference Example 1) alone or (b) L3-1Y and sorafenib. For the single treatment of L3-1Y alone, the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL. For the co-treatment of L3-1Y and sorafenib, the concentration of sorafenib was fixed at 4 μM, and the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL.

At 72 hours after the single treatment or the co-treatment, 100 μL of CellTiter Glo solution (Promega, G7572) was added to each well, and left for 30 minutes at room temperature. The cell number was counted by emission signal, and the emission signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer).

The obtained results are shown in FIG. 3. As shown in FIG. 3, in the HT29 cell line, the cell growth inhibiting effect is not observed when L3-1Y is administered alone; however, it is observed when L3-1Y and sorafenib are co-administered. Considering that the single treatment of L3-1Y leads to no cell growth inhibiting effect on HT29 cells, the synergistic effect by the co-treatment of L3-1Y and sorafenib is considerably beyond the expected scope and shows the acquirement of an anticancer effect on the cancer cells on which L3-1Y does not exhibit anticancer effect when administered alone. These results indicate that by the co-treatment of L3-1Y and sorafenib, the cancer cell growth inhibiting effect of L3-1Y on HT29 cells can be acquired, whereby the coverage of L3-1Y can be extended to cancer cells such as colon cancer cells on which L3-1Y does not exhibit an anticancer effect when administered alone.

Example 4 Verification of the Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Breast Cancer Cell Line

The effect of co-administration of sorafenib and anti-c-Met antibody L3-1Y prepared in Reference Example 1 was verified in a breast cancer cell line (HCC1806 cell line).

In particular, RPMI1640 medium (GIBCO) containing 10% FBS was added in a 96 well plate. HT29 colon cancer cells (ATCC, HTB-38) were seeded thereon in the amount of 5,000 cells/well, and incubated overnight at 37° C. After 24 hours (the next day), the incubated cells were treated with (a) L3-1Y (Reference Example 1) alone or (b) L3-1Y and sorafenib. For the single treatment of L3-1Y alone, the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL., For the co-treatment of L3-1Y and sorafenib, the concentration of sorafenib was fixed at 3 μM, and the concentration of L3-1Y was 0 μg/mL, 0.016 μg/mL, 0.08 μg/mL, 0.4 μg/mL, or 2 μg/mL.

At 72 hours after the single treatment or the co-treatment, 100 μL of CellTiter Glo solution (Promega, G7572) was added to each well, and left for 30 minutes at room temperature. The cell number was counted by emission signal, and the emission signal was recorded using Envision 2104 Multi-label Reader (Perkin Elmer).

The obtained results are shown in FIG. 4. As shown in FIG. 4, in the HCC1806 cell line, the cell death effect is not observed when L3-1Y is administered alone; however, the cell growth inhibiting effect depending on the concentration of L3-1Y is clearly observed when L3-1Y and sorafenib are co-administered. Considering that the single administration of L3-1Y leads to no cell growth inhibiting effect on HCC1806 cells, the synergistic effect caused by the co-treatment of L3-1Y and sorafenib is considerably beyond the expected scope and shows the acquirement of an anticancer effect on the cancer cells on which L3-1Y does not exhibit an anticancer effect when administered alone. These results indicate that by the co-treatment of L3-1Y and sorafenib, the cancer cell growth inhibiting effect of L3-1Y on HCC1806 cells can be acquired, whereby the coverage of L3-1Y can be extended to cancer cells such as breast cancer cells on which L3-1Y does not exhibit anticancer effect when administered alone.

Example 5 Verification of the Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Tumor Xenograft Model of a Gastric Cancer Cell Line

To verify the in vivo effect of the co-administration on tumor growth, a tumor xenograft model using male 5-6 week-old BALB/c nude mice (Pharmalegacy, China) was prepared. The mice were adapted at least one week before inoculation of tumor cells.

Then, the mice were anesthetized with 1-2% isofurane, and MKN45 gastric cancer cells (JCRB, JCRB0254) were subcutaneously injected into the right flank of the mice in the amount of 5×10⁶ cells per mouse. 7 days after, when the average size of the tumor reaches 50 mm³ or more, the mice were grouped as follows: a group treated with sorafenib (3.3 mg/kg, P.O., once a day), a group treated with L3-1Y (1 mg/kg, I.V., once a week), a group co-treated with L3-1Y (1 mg/kg, I.V., once a week) and sorafenib (3.3 mg/kg, P.O., once a day), and a group (control) treated with vehicle (PBS 0.2 ml, I.V., once a week). Each group consisted of 10 mice.

The experiment was performed for 4 weeks in total, and tumor size and body weight of the mice were measured twice a week. The tumor volume (V) was calculated by the following formula:

V (mm³)={length of long axis (mm)×(length of short axis (mm))²}/2.

The obtained results are illustrated in FIG. 5 and Table 3.

TABLE 3 Tumor Inhibition Rate at 21^(st) day (%; MKN45 compared to vehicle-treated group (0%)) Vehicle 0.0 Sorafenib 24.7 L3-1Y 36.2 Sorafenib + L3-1Y 55.9

Table 3 shows the tumor inhibition rate (%), which is calculated by comparing tumor size of each group at the 21^(st) day after treatment with that of the vehicle-treated group, wherein the tumor inhibition rate of the vehicle-treated group is expressed as 0%. As shown in FIG. 5 and Table 3, the group co-treated with L3-1Y and sorafenib shows a significantly increased tumor inhibiting effect compared with the groups treated with L3-1Y or sorafenib only.

Example 6 Verification of the Cancer Cell Growth Inhibiting Effect by Co-Administration of Sorafenib and L3-1Y in a Tumor Xenograft Model of a Colon Cancer Cell Line

To verify the in vivo effect of the co-administration on tumor growth, a tumor xenograft model using male 5-6 week-old BALB/c nude mice (Pharmalegacy, China) was prepared. The mice were adapted at least one week before inoculation of tumor cells.

Then, the mice were anesthetized with 1-2% isofurane, and HT29 colon cancer cells I(ATCC, HTB-38) were subcutaneously injected into the right flank of the mice at the amount of 5×10⁶ cells per mouse. 7 days after, when the average size of the tumor reaches 50 mm³ or more, the mice were grouped as follows: a group treated with sorafenib (3.3 mg/kg, P.O., once a day), a group treated with L3-1Y (1 mg/kg, I.V., once a week), a group co-treated with L3-1Y (1 mg/kg, I.V., once a week) and sorafenib (3.3 mg/kg, P.O., once a day), and a group (control) treated with vehicle (PBS 0.2 ml, I.V., once a week).

The experiment was performed for 4 weeks in total, and tumor size and body weight of the mice were measured twice a week. The tumor volume (V) was calculated by the following formula:

V (mm³)={length of long axis (mm)×(length of short axis (mm))²}/2.

The obtained results are illustrated in FIG. 6 and Table 4.

TABLE 4 Tumor Inhibition Rate at 21^(st) day (%; HT29 compared to vehicle-treated group (0%)) Vehicle 0.0 Sorafenib 22.1 L3-1Y −0.9 Sorafenib + L3-1Y 36.2

Table 4 shows the tumor inhibition rate (%), which is calculated by comparing the tumor size of each group at the 21^(st) day after treatment with that of the vehicle-treated group, wherein the tumor inhibition rate of the vehicle-treated group is expressed as 0%. As shown in FIG. 6 and Table 4, the group co-treated with L3-1Y and sorafenib shows a significantly increased tumor inhibiting effect compared with the groups treated with L3-1Y or sorafenib only. In particular, when L3-1Y was treated alone, no tumor inhibiting effect is observed.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method for prevention or treatment of a cancer, comprising co-administering (a) sorafenib and (b) an anti-c-Met antibody or antigen-binding fragment thereof to a subject in need thereof, wherein the anti-c-Met antibody or the antigen-binding fragment thereof specifically binds to an epitope comprising 5 or more contiguous amino acids within the SEMA domain of c-Met protein, thereby preventing or treating cancer in the subject.
 2. The method of claim 1, wherein sorafenib and the anti-c-Met antibody or the antigen-binding fragment are administered simultaneously or sequentially in any order.
 3. The method according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof specifically binds to an epitope comprising 5 to 19 contiguous amino acids of SEQ ID NO: 71 including the amino acid sequence of SEQ ID NO:
 73. 4. The method according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof specifically binds to an epitope comprising an amino acid sequence of SEQ ID NO: 71, 72, or
 73. 5. The method according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises: (i) at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence comprising 8-19 consecutive amino acids within the amino acid sequence of SEQ ID NO: 2 comprising amino acid residues from the 3^(rd) to 10^(th) positions of the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutive amino acids within the amino acid sequence of SEQ ID NO: 85 comprising amino acid residues from the 1^(st) to 6^(th) positions of the amino acid sequence of SEQ ID NO: 85, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region; (ii) at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence comprising 9-17 consecutive amino acids within the amino acid sequence of SEQ ID NO: 89 comprising amino acid residues from the 1^(st) to 9^(th) positions of the amino acid sequence of SEQ ID NO: 89, or a light chain variable region comprising the at least one light chain complementarity determining region; (iii) a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or (iv) a combination of the heavy chain variable region and the light chain variable region.
 6. The method according to claim 5, wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO: 26, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 85, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 106, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, or SEQ ID NO:
 89. 7. The method according to claim 5, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, and 94, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, and
 107. 8. The method according to claim 5, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 62, the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64, the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66, or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66; and a light chain comprising the amino acid sequence of SEQ ID NO: 68, the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70, the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70, or the amino acid sequence of SEQ ID NO:
 108. 9. The method according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises a light chain complementarity determining region comprising the amino acid sequence of SEQ ID NO: 106, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107, or a light chain comprising the amino acid sequence of SEQ ID NO:
 108. 10. The method according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66 and a light chain comprising the sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO:
 68. 11. The method according to claim 1, wherein the anti-c-Met antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid sequence of SEQ ID NO:
 110. 12. The method according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)₂, scFvFc, Fab, Fab′ and F(ab′)₂ derived from the anti-c-Met antibody.
 13. The method according to claim 1, wherein the cancer is at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer. 